Injection Of Cholera Toxin B Subunit Research Articles

Studies of the central neural pathways to the stomach and Zusanli (ST36).

The purpose of this morphological study was to investigate the relation between the meridian, meridian points and viscera using neuroanatomical tracers. The common locations of the spinal cord and brain projecting to the stomach and Zusanli were observed following injection of CTB (cholera toxin B subunit) and pseudorabies viruses (PRV-Ba, Bartha strain and PRV-Ba-Gal, galactosidase insertion) into the stomach and Zusanli (ST36). After 4-5 days of survival following injection into twelve rats, they were perfused, and their spinal cords and brains were frozen sectioned (30 microm). These sections were stained by X-gal histochemical, CTB and PRV-Ba immunohistochemical staining methods, and examined with the light microscope. The results were as follows: Commonly labeled medulla oblongata regions were dorsal motor nucleus of vagus nerve (DMV), nucleus tractus solitarius (NTS) and area postrema (AP) following injection of CTB and PRV-Ba-Gal into stomach and Zusanli, respectively. In the spinal cord, commonly labeled neurons were found in thoracic, lumbar and sacral spinal segments. Densely labeled areas were found in lamina IV, V, VII (intermediolateral nucleus) and X of the spinal cord. In the brain, commonly labeled neurons were found in the Al noradrenalin cells/Cl adrenalin cells/caudoventrolateral reticular nucleus, dorsal motor nucleus of vagus nerve, nucleus tractus solitarius, area postrema, raphe obscurus nucleus, raphe pallidus nucleus, raphe magnus nucleus, gigantocellular nucleus, locus coeruleus, parabrachial nucleus, Kolliker-Fuse nucleus, A5 cell group, central gray matter, paraventricular hypothalamic nucleus, lateral hypothalamic nucleus, retrochiasmatic hypothalamic nucleus, bed nucleus of stria terminalis and amygdaloid nucleus. Thus central autonomic center project both to the stomach and Zusanli. These morphological results suggest that there is a commonality of CNS cell groups in brain controlling stomach (viscera) and Zusanli (limb).

Neonatal whisker clipping alters intracortical, but not thalamocortical projections, in rat barrel cortex.

Retrograde axonal transport of cholera toxin B subunit (CTB) was used to compare the development of intracortical and thalamocortical connections in normal rats with those in rats in which all of the whiskers were trimmed continuously from birth. In normal animals, injections of CTB into a single barrel column resulted in an asymmetrical labeling of cells that were distributed preferentially within columns related to the same row in which the injection was placed. This anisotropy in the patterns of intracortical connections was not observed in whisker-clipped animals. In these animals, there was a significant reduction in the mean number of labeled cells in the infragranular layers, and labeled cells were distributed symmetrically around the injection site. The same injections of CTB also labeled thalamocortical neurons in the ventrobasal thalamus. Analysis of the distribution of these cells revealed that, in both control and experimental animals, the vast majority of labeled cells were restricted to a homologous (i.e., corresponding to the injected cortical barrel) thalamic barreloid. These findings indicate that manipulations of sensory experience alter patterns of intracortical, but not thalamocortical, connections.

Organization of the nigro-tecto-bulbar pathway to the parvicellular reticular formation: a light- and electron-microscopic study in the rat.

We examined a nigro-tecto-bulbar pathway to the parvicellular reticular formation (RFp), where many premotor neurons for orofacial motor nuclei are known to be distributed, by using a combined anterograde and retrograde tracing method. After contralateral injections of biotinylated dextranamine (BDA) into the dorsolateral part of the substantia nigra (SNr) and cholera toxin B subunit (CTb) into the RFp, overlapping distributions of BDA-labeled terminals and CTb-labeled neuronal cell bodies were found in the lateralmost part of the superior colliculus (SC) ipsilateral or contralateral to the site of BDA injection or CTb injection, respectively. After contralateral injections of BDA into the SNr and horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) injection into the RFp, ipsilateral labeled axon terminals with BDA were found to make symmetrical synaptic contacts with the somata and dendrites of contralateral labeled neurons with WGA-HRP in the lateralmost part of the SC. Furthermore, we demonstrated that BDA-labeled axon terminals were immunoreactive for GABA, by using the anterograde tracing method combined with immunohistochemistry for GABA. Thus, GABA-like immunoreactive fibers originating from the dorsolateral part of the SNr make monosynaptic contacts with the tectal neurons sending their axons to the RFp.

Anterograde tracing of retinohypothalamic afferents with Fluoro-Gold

The anterograde neuronal tracing properties of Fluoro-Gold (FG) were characterized in this study by its ability to label the retinohypothalamic tract (RHT) upon pressure injection of the substance into the vitrous body of the eye in the Djungarian hamster, Phodopus sungorus. Tracing was compared to the anterograde neuronal transport of cholera toxin B subunit (CTB), Fast blue (FB), Phaseolus vulgaris leucoagglutinin (PHA-L) and biocytin. After survival times that ranged from 24 h to 4 weeks, a major projection was found to the bilateral hypothalamic suprachiasmatic nuclei (SCN). Labeling was also found in the anterior medial preoptic nucleus and, in relatively sparse amounts, in the lateral geniculate nucleus, superior colliculus and lateral habenular nucleus. Similar results were obtained upon injection of CTB or FB, respectively, into the eye, whereas the application of PHA-L or biocytin did not label retinal afferents. The combined injection of FG and CTB or FB into the same eye labeled retino-afferent fibers only when FG was applied three days before the injection of the other tracers. Retrogradely labelled neurons were seen occasionally in the hypothalamus which may provide a sparse retinopetal projection. Additional experiments combining FG tracing and the immunofluorescent detection of the neuropeptides substance P (SP) or vasoactive intestinal polypeptide (VIP) in the SCN showed that FG-containing punctae were accumulated in the vicinity of immunoreactive cell bodies. Our data demonstrate that FG may be used as an anterograde axonal tracer of the retinohypothalamic pathway.

Lower brainstem catecholamine afferents to the rat dorsal raphe nucleus.

A large body of data suggests that the activation of alpha 1 receptors by a tonic noradrenergic input might be responsible for the tonic discharge of the serotonergic neurons of the dorsal raphe nucleus (DRN). To test this hypothesis, it was necessary to determine the origin of the noradrenergic and adrenergic innervation of these neurons. For this purpose, we combined small iontophoretic injections of the sensitive retrograde tracer cholera toxin b subunit (CTb) in the different subdivisions of the DRN with tyrosine hydroxylase immunohistochemistry. After CTb injections in the ventral or dorsal parts of the central DRN, a small number of double-labeled cells was observed in the locus coeruleus (A6 noradrenergic cell group), the A5 noradrenergic group, the dorsomedial medulla (C3 adrenergic cell group), and the lateral paragigantocellular nucleus (C1 adrenergic cell group). After CTb injections in the lateral wings or the dorsal part of the rostral DRN, a similar number of double-labeled cells was seen in C3. Slightly more double-labeled cells were seen in A6 and A5. In addition, a substantial to large number of double-labeled cells appeared in C1, the commissural part of the nucleus of the solitary tract (A2 noradrenergic cell group) and the caudoventrolateral medulla (A1 noradrenergic cell group). These results indicate that the noradrenergic and adrenergic inputs to the DRN arise from all the catecholaminergic cell groups of the lower brainstem except the A7 noradrenergic group. They further reveal the existence of a topographical organization of these afferents to the different subdivisions of the DRN.

Reciprocal and topographic connections between the piriform and prefrontal cortices in the rat: a tracing study using the B subunit of the cholera toxin.

In the present study, the reciprocal connections between the piriform cortex and the prefrontal areas are described on the basis of experiments using the anterograde and the retrograde transport of the cholera toxin B subunit (CTb). Following CTb injections placed in the anterior part of the piriform cortex, retrogradely labeled cells and anterogradely labeled fibers were mainly found in the ventrolateral and lateral orbital areas as well as in the anterior part of the agranular insular cortex. Following injections placed in the posterior part of the piriform cortex, the CTb labeling was primarily observed in the infralimbic area and the posterior part of the agranular insular cortex. Thus, we described a topographical organization of the direct reciprocal connections between the anterior and the posterior parts of the piriform cortex parts and some prefrontal areas. This could support a differential modulation of the olfactory processing along the rostrocaudal dimension of the piriform cortex.

VIP-like immunoreactive projections from the dorsal raphe and caudal linear raphe nuclei to the bed nucleus of the stria terminalis demonstrated by a double immunohistochemical method in the rat

To localize the vasoactive intestinal polypeptide (VIP)-like immunoreactive neurons at the origin of the plexuses of VIP-immunoreactive fibers found in the bed nucleus of the stria terminalis (BNST), we combined iontophoretic injections of the retrograde tracer cholera toxin B-subunit (CTb) with VIP immunocytochemistry. Following CTb injections in the BNST, 2 groups of retrogradely labeled neurons immunoreactive to VIP were detected: (1) a dorso-caudal group localized in the dorsal part of the dorsal raphe nucleus and the adjacent ventro-lateral part of the periaqueductal gray; and (2) a rostral group in the caudal linear raphe nucleus. No double-labeled cells were observed in other nuclei including those of the amygdala and the supramammillary nucleus.

Evidence that cholera toxin B subunit (CTb) can be avidly taken up and transported by fibers of passage

It has been reported that cholera toxin B subunit (CTb) is a sensitive neuronal tracer with unique features. However, the possible uptake of CTb by non-terminal fibers passing through the injection site has not been examined thoroughly. In the present study, small iontophoretic injections (current = + 2 μA) of CTb were made in the olivocerebellar pathway in the rat ventrolateral medulla. A large number of retrogradely labeled neurons were seen in the contralateral inferior olive. In addition, prominent anterogradely labeled climbing fibers/terminals were found in the cerebellum ipsilateral to the injection site. This study, in contrast to previous report(s), indicates that CTb can be taken up avidly by fibers of passage and transported both anterogradely and retrogradely.

Serotonergic and non-serotonergic projections from the raphe nuclei to the piriform cortex in the rat: a cholera toxin B subunit (CTb) and 5-HT immunohistochemical study

Retrograde axonal transport of the cholera toxin B subunit (CTb) was combined with 5-HT immunohistochemistry to determine the origin of the serotonergic innervation of the piriform cortex (PC) in the rat. After iontophoretic CTb injections in the PC, a substantial number of retrogradely labeled cells were found in the middle and medio-ventral part of the dorsal raphe nucleus (RD). A few retrogradely labeled cells were also observed in the median raphe nucleus (MnR) and the B9 serotonergic cell groups. Following CTb and 5-HT immunohistochemistry on the same sections, double-labeled cells were observed in the RD, MnR and B9 groups. In the RD, 30% of CTb stained cells were immunoreactive to 5-HT. After colchicine or nialamide (a monoamine oxidase inhibitor) pretreatment the percentage of these double-labeled cells reached 70%. These results indicate that both 5-HT and non-5-HT neurons in the RD innervate the PC and that the percentage of double-labeled cells is influenced by drug pretreatment. To determine the terminal fields of the RD efferent fibers in the PC, injections of the anterograde tracer PHA-L were also performed. Analysis of the fiber distribution in the PC further revealed some medio-lateral and antero-posterior differences.

Afferents to the nucleus reticularis parvicellularis of the cat medulla oblongata: a tract-tracing study with cholera toxin B subunit.

The aim of this study was to examine anatomical evidence in cats of whether the nucleus reticularis parvicellularis (Pc) is part of the circuit responsible for the inhibition of brainstem motoneurons during paradoxical sleep. For this purpose, we made iontophoretic injections of the retrograde and anterograde tracer cholera toxin B subunit (CTb) in the Pc. After CTb injections in the Pc, a large number of retrogradely labeled neurons were seen in the central nucleus of the amygdala, the lateral part of the bed nucleus of the stria terminalis, the posterior hypothalamic areas, the mesencephalic reticular formation, the nucleus locus subcoeruleus, the nucleus pontis caudalis, other portions of the Pc, the nucleus reticularis dorsalis, the trigeminal sensory complex, and the nucleus of the solitary tract. We further found that the Pc receives 1) serotoninergic afferents from the raphe dorsalis, magnus, and obscurus nuclei; 2) noradrenergic inputs from the dorsolateral pontine tegmentum; 3) cholinergic afferents from the lateral medullary reticular formation; 4) substance P-like afferents from the central nucleus of the amygdala, bed nucleus of the stria terminalis, periaqueductal gray, and nucleus of the solitary tract; and 5) methionine-enkephalin-like projections from the periaqueductal gray, the nucleus of the solitary tract, the lateral pontine and medullary reticular formation, and the spinal trigeminal nucleus. We further found that the Pc do not receive afferents from brainstem structures responsible for muscle atonia, such as the ventromedial medulla and the dorsomedial pontine tegmentum, and therefore may not be part of the circuit inhibiting the brainstem motoneurons during paradoxical sleep.

Brainstem dopaminergic, cholinergic and serotoninergic afferents to the pallidum in the squirrel monkey

The retrograde tracer cholera toxin B subunit (CTb) was used in combination with immunohistochemistry for tyrosine hydroxylase (TH), calbindin D-28k (CaBP), choline acetyltransferase (ChAT) and 5-hydroxytryptamine (5-HT) to determine the distribution and relative proportion of brainstem chemospecific neurons that project to the pallidum in the squirrel monkey ( Saimiri sciureus). Large injections of CTb involving both pallidal segments produce numerous retrogradely labeled neurons in the substantia nigra (SN), the pedunculopontine tegmental nucleus (PPN) and the dorsal raphe nucleus (DR). Labeled neurons are distributed uniformly in SN with a slight numerical increase at the junction between the pars compacta (SNc) and the ventral tegmental area (VTA). Retrogradely labeled neurons abound also in PPN, principally in its pars dissipata, whereas other CTb-labeled cells are scattered throughout the rostrocaudal extent of DR. After CTb injection involving specifically the internal pallidal segment (GPi), the same pattern of cell distribution is found in SN, PPN and DR, except that the number of retrogradely labeled cells is lower than after large pallidal complex injections. Approximately 70% of all CTb-labeled neurons in SNc-VTA complex display TH immunoreactivity, whereas 20% are immunoreactive for CaBP. About 39% of all retrogradely labeled neurons in PPN are immunoreactive for ChAT, whereas approximately 38% of the labeled neurons in DR display 5-HT immunoreactivity. Following CTb injection in the external pallidal segment (GPe), the number of labeled cells is much smaller than after GPi injection. The majority of CTb-labeled cells in SNc-VTA complex are located in the lateral half of SNc and approximately 93% of these neurons display TH immunoreactivity compared to 10% that are immunoreactive for CaBP; very few CTb-labeled cells occur in PPN. Retrogradely labeled cells in DR are located more laterally than those that projects to the GPi and about 25% of them are immunoreactive for 5-HT. These results suggest that, in addition to their action at striatal and/or nigral levels, the brainstem dopaminergic, cholinergic and serotoninergic neurons influence the output of the primate basal ganglia by acting directly upon GPi neurons.